Human T-cell leukemia virus (HTLV-1) infects about 20 million individuals worldwide and is the etiological agent of an adult T-cell leukemia/lymphoma (ATLL), and can also result in an inflammatory disease syndrome called HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). Prevalence rates for HTLV-1 infection in the general population are greater than 1% in the Caribbean Basin, Central Africa, and South Japan. HTLV-1 is notorious for being difficult to study in cell culture, which ha prohibited a rigorous analysis of how these viruses replicate in cells, including the steps involved in retrovirus assembly. The details for how retrovirus particle assembly occurs are poorly understood even for other more tractable retroviral systems. Using a tractable model system, state-of-the-art biophysical approaches, and an interdisciplinary research team, we have made novel observations that form the basis for this proposal. In this application, we propose to investigate questions related to HTLV-1 particle size, Gag stoichiometry in particles, and HTLV-1 Gag interactions in living cells using multiple experimental approaches. In particular, we will apply cryo-electron microscopy/tomography (cryo-EM/ET), total internal reflection fluorescence (TIRF) microscopy, and the novel single-molecule technology of fluorescence fluctuation spectroscopy (FFS) to investigate questions related to 1) particle size and Gag stoichiometry, 2) Gag targeting to membrane, and, 3) HTLV-1 particle biogenesis. The results from these proposed studies should provide further insight into fundamental aspects of HTLV-1 and retrovirus particle assembly, which may aid in developing therapeutics.